The overall goal of this application is to measure the cell-mediated cytolytic reactivities of patient-derived effector cell populations against molecularly-defined tumor-associated antigens (TAA) in order to identify specific targets for immunotherapeutic strategies. This approach, which brings together established investigators from the fields of surgical oncology, molecular biology, and tumor immunology, will be based largely on both recently developed vector-based targeting techniques developed for the measurement of anti-viral cytotoxicities as well as new refinements in effector cell capture and ex vivo expansion technologies. A panel of specific poxvirus/TAA vectors will be constructed and used to target autologous, EBV-transformed patient B-cell lines for the measurement of anti-tumor cytolytic T-cell (CTL) and antibody-dependent cellular cytotoxicities (ADCC). These constructs will likewise be utilized in specific in vitro stimulation strategies aimed at expansion of low-frequency CTL reactivities present in peripheral blood mononuclear cells (PBMC) and/or tumor-infiltrating lymphocytes (TIL) obtained from tumorbearing patients. These efforts will concentrate on 3 specific human cancers--namely, 1) carcinoma of the breast, 2) pancreatic carcinoma, and 3) malignant melanoma, as putative TAA targets have been identified in each of these tumor systems. Primary focus will be directed toward mutated forms of the tumor suppressor gene p53 in breast cancer, based on observations by two of the co-investigators that a subset of patients have high anti-p53 antibody titers. Such patients will be extensively studied in efforts to determine whether the immunogenicity of p53 extends into the cellular arm of the immune response. Similar studies will be performed in patients with diagnosed pancreatic carcinomas in which greater than 60% of tumors share conserved mutations in codons 12 of K-ras. The cellular cytolytic response against the product(s) of the p97 onc-gene will be measured in a cohort of malignant melanoma patients entered on ongoing clinical protocols at Duke involving repeated immunization with lysates of allogeneic tumor cell lines. Lastly, a developmental strategy aimed at identification of new TAA targets for patient CTL will be attempted utilizing a library of recombinant poxvirus expression vectors. Collectively, these studies will reveal whether conserved tumor-associated antigens can serve as CTL targets for patient effector cell populations and, ultimately, as targets for interventive immunotherapeutic modalities.